Retroviruses pose a threat to human health by infecting somatic cells, but retroviruses have also been infecting our mammalian ancestors for millions of years, accumulating in the germ-line as ERVs that account for nearly 10% of our genomic DNA. My laboratory studies ERVs from two perspectives: 1) as parasites that must be kept in check by the host to prevent widespread viral activation and 2) as symbionts that can be co-opted by the host for evolutionary advantage. Our objective is to understand how the host has adapted recognition machinery to establish stable epigenetic silencing of ERVs, how ERVs sometimes evade these silencing mechanisms, and how these evasive activities have lead to host co-option of viral regulatory sequences that may have contributed to evolution of mammals. We hypothesize that the rapidly diversifying KRAB-ZFP family plays a critical role in the recognition and silencing of ERVs. Kruppel associated box zinc finger proteins (KRAB-ZFPs) have emerged as candidates that recognize ERVs KRAB-ZFPs are rapidly evolving transcriptional repressors that emerged in tetrapods. They make up the largest family of transcription factors in mammals (estimated to be 200-300 in mice and humans) 1. Each species has its own unique repertoire of KRAB-ZFPs, with a small number shared with closely related species and a larger fraction specific to each species. Despite their abundance, little is known about their physiological functions. KRAB-ZFPs consist of an N-terminal KRAB domain that binds the co-repressor KAP12 and a variable number of C-terminal C2H2 zinc finger domains that mediate sequence-specific DNA binding. KAP1 directly interacts with the KRAB domain3, which recruits the histone methyltransferase (HMT) SETDB1 and heterochromatin protein 1 (HP1) to initiate heterochromatic silencing4,5. Several lines of evidence point to a role for the KRAB-ZFP family in ERV silencing. First, the number of C2H2 zinc finger genes in mammals correlates with the number of ERVs 6. Second, the KRAB-ZFP protein ZFP809 was isolated based on its ability to bind to the primer binding site for proline tRNA (PBSPro) of murine leukemia virus (MuLV) 7. Third, deletion of the KRAB-ZFP co-repressors Trim28 or Setdb1 leads to activation of many ERVs 8,9. Thus we have begun a systematic interrogation of KRAB-ZFP function as a potential adaptive repression system against ERVs. We focused on ZFP809 as a likely ERV-suppressing KRAB-ZFP since it was originally identified as part of a repression complex that recognizes infectious MuLV via direct binding to the 18 nt Primer Binding Site for Proline (PBSpro) sequence7,10. We hypothesized that ZFP809 might function in vivo to repress other ERVs that utilized the PBSpro. Using ChIP-seq of epitope tagged ZFP809 in ESCs and embryonic carcinoma (EC) cells, we determined that ZFP809 bound to several sub-classes of ERV elements via the PBSpro. We generated Zfp809 knockout mice to determine whether ZFP809 was required for VL30pro silencing. We found that Zfp809 knockout tissues displayed high levels of VL30pro elements and that the targeted elements display an epigenetic shift from repressive epigenetic marks (H3K9me3 and CpG methylation) to active marks (H3K9Ac and CpG hypo-methylation). ZFP809-mediated repression extended to a handful of genes that contained adjacent VL30pro integrations. Furthermore, using a combination of conditional alleles and rescue experiments, we determined that ZFP809 activity was required in development to initiate silencing, but not in somatic cells to maintain silencing. These studies provided the first demonstration for the in vivo requirement of a KRAB-ZFP in the recognition and silencing of ERVs. As a follow-up to our studies on ZFP809, we have begun a systematic analysis of KRAB-ZFPs using a medium throughput ChIP-seq screen and functional genomics of KRAB-ZFP clusters and individual KRAB-ZFP genes. Our ChIP-seq data demonstrates that the majority of recently evolved KRAB-ZFP genes interact with and repress distinct and partially overlapping ERV targets. This is supported by a recent knockout mouse line lacking 17 KRAB-ZFPs (generated with CRISPR/Cas9 engineering) that displays an ERV reactivation phenotype. Although our data shows that many KRAB-ZFPs repress ERVs, we also found that more ancient KRAB-ZFPs that emerged in a human/mouse common ancestor do not bind and repress ERVs. One of these KRAB-ZFPs plays an important role in silencing a key developmental gene that may have played a critical role in the onset of viviparity in mammals. 1 Urrutia, R. KRAB-containing zinc-finger repressor proteins. Genome Biol 4, 231, doi:10.1186/gb-2003-4-10-231 (2003). 2 Friedman, J. R. et al. KAP-1, a novel corepressor for the highly conserved KRAB repression domain. Genes Dev 10, 2067-2078 (1996). 3 Peng, H. et al. Biochemical analysis of the Kruppel-associated box (KRAB) transcriptional repression domain. J Biol Chem 275, 18000-18010, doi:10.1074/jbc.M001499200 (2000). 4 Ryan, R. F. et al. KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Kruppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing. Mol Cell Biol 19, 4366-4378 (1999). 5 Schultz, D. C., Ayyanathan, K., Negorev, D., Maul, G. G. & Rauscher, F. J., 3rd. SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev 16, 919-932, doi:10.1101/gad.973302 (2002). 6 Thomas, J. H. & Schneider, S. Coevolution of retroelements and tandem zinc finger genes. Genome Res 21, 1800-1812, doi:10.1101/gr.121749.111 (2011). 7 Wolf, D. & Goff, S. P. Embryonic stem cells use ZFP809 to silence retroviral DNAs. Nature 458, 1201-1204, doi:10.1038/nature07844 (2009). 8 Rowe, H. M. et al. KAP1 controls endogenous retroviruses in embryonic stem cells. Nature 463, 237-240, doi:10.1038/nature08674 (2010). 9 Matsui, T. et al. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature 464, 927-931, doi:10.1038/nature08858 (2010). 10 Wolf, D. & Goff, S. P. TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells. Cell 131, 46-57, doi:10.1016/j.cell.2007.07.026 (2007).